Metabolic labeling of glycans with isotopic glucose for quantitative glycomics in yeast.

Changes in glycan levels could directly affect the biochemical properties of glycoproteins and thus influence their physiological functions. In order to decode the correlation of glycan prevalence with their physiological contribution, many mass spectrometry (MS) and stable isotope labeling-based methods have been developed for the relative quantification of glycans. In this study, we expand the quantitative glycomic toolbox with the addition of optimized Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose (MILPIG) approach in baker's yeast (Saccharomyces cerevisiae). We demonstrate that culturing baker's yeast in the presence of carbon-13 labeled glucose (1-13C1) leads to effective incorporation of carbon-13 to both N-linked and O-linked glycans. We established that metabolic incorporation of isotope-labeled glucose at a concentration of 5 mg/mL for three days is required for an accurate quantitative analysis with optimal isotopic cluster distribution of glycans. To validate the robustness of the method, we performed the analysis by 1:1 mixing of normal and isotope-labeled glycans, and obtained excellent linear calibration curves from various analytes. Finally, we quantitated the inhibitory effect of tunicamycin, a N-linked glycosylation inhibitor, to glycan expression profile in yeast.

Involvement of antioxidant defense system in solvent tolerance of Pseudomonas putida BCNU 106.

The highly solvent-tolerant bacterium Pseudomonas sp. BCNU 106 was investigated to elucidate the solvent tolerance under specific culture conditions with the presence of solvents and its adaptive mechanisms to those conditions with reference to the antioxidant system. When exposed to 10% toluene, Pseudomonas sp. BCNU 106 increased the generation of reactive oxygen species assessed by monitoring the oxidation of 2',7'-dichlorofluorescein. Typical antioxidant enzymes (viz. catalase, superoxide dismutase, and glutathione reductase) showed increased activity with prolonged incubation in 10% toluene. In addition, the levels of these antioxidant proteins were higher during exposure to 10% toluene than in toluene-free condition. The present study indicates that antioxidant defense activity is one of the adaptive and protective mechanisms developed to avoid the deleterious damage of organic solvents, especially toluene.

Changes in antioxidant defense systems by 2,2',5,5'-tetrachlorobiphenyl exposure in neuronal SK-N-MC cells.

Polychlorinated biphenyls (PCBs) are known to alter the mammalian antioxidant defense system. To determine whether similar detoxification processes are activated in human neuronal cells, we investigated activities of antioxidant enzymes and the glutathione status (i.e., the levels of reduced and oxidized glutathione, GSH and GSSG) in human neuronal SK-N-MC cells exposed to 2,2',5,5'-tetrachlorobiphenyl (PCB 52). Upon PCB 52 treatment, time- and concentration-dependent inhibitions of cell viability were observed. PCB 52 did not affect GSH contents upon increasing the concentration up to 15 microg/ml, but significant depletions in GSH were observed at the concentrations of 20 and 25 microg/ml. PCB 52 exposure increased GSSG levels in the SK-N-MC cells, while GSH levels were decreased, and these changes naturally modified the GSSG/GSH ratios. Cytosolic glutathione S-transferase (GST) activity with 1-chloro-2,4-dinitrobenzene as substrate was enhanced by two-fold in neuronal cells after exposure to PCB 52 versus controls. In contrast, neuronal cells showed a sustained decrease in glutathione peroxidase activity with increasing concentrations of PCB 52, and a sustained decrease in Cu/Zn-superoxide dismutase (SOD) activity with increasing concentrations of PCB 52. Catalase activity was increased until 12 h after exposure to PCB 52, but was decreased 24 h after exposure. Overall, these results imply a major effect of PCB 52 on GSH status and upon the activities of antioxidant enzymes in human neuronal SK-N-MC cells, and upon the overall process of detoxification in human neuronal cells.

IKKgamma inhibits activation of NF-kappaB by NIK.

IKKgamma is a component of the IKK complex, which regulates NF-kappaB activity. To investigate the role of IKKgamma, we expressed wild type IKKgamma containing 412 amino acids, and deletion mutants containing residues 1-312 and 101-412, using murine IKKgamma cDNA. In a co-transfection assay with a CAT reporter plasmid, NIK activated NF-kappaB-dependent gene expression approximately two fold and this expression was inhibited by co-transfection of a wild type IKKgamma expression plasmid. In binding assays IKKgamma inhibited the association of IkappaBalpha with IKKbeta and the subsequent phosphorylation of IkappaBalpha that is activated by NIK. Inhibition by IKKgamma also occurred in an assay with a dominant negative mutant of NIK but not with a C-terminal deletion mutant of IKKgamma, indicating that the C-terminal 100 amino acids of IKKgamma are important for negative regulation of NF-kappaB activation. In addition, the interaction of IKKbeta with IKKgamma was inhibited by co-transfection with a NIK expression plasmid. Our results suggest that overexpression of IKKgamma inhibits activation of NF-kappaB by NIK by competing with NIK for interaction with IKKbeta.

Protective effect of ginseng extract against apoptotic cell death induced by 2,2',5,5'-tetrachlorobiphenyl in neuronal SK-N-MC cells.

Oxidative stress plays an important role in the pathological processes of neurodegenerative diseases. Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic. 2,2',5,5'-Tetrachlorobiphenyl (PCB 52) induces apoptotic death in human neuronal SK-N-MC cells, as demonstrated by gel electrophoresis, which demonstrates the proteolytic cleavage of beta-catenin and poly(ADP-ribose) polymerase (PARP) and the characteristic ladder patterns of DNA fragmentation. In the present study, we investigated whether Panax ginseng extract protect human neuronal SK-N-MC cells from PCB 52-induced apoptosis. The addition of 500 microg/ml of ginseng extract to a culture medium significantly protected neuronal cell from the apoptosis mediated by PCB 52 and remarkably attenuated lipid peroxidation, the generation of reactive oxygen species, and DNA fragmentation, and markedly reduced the PCB 52 induced proteolytic cleavage of beta-catenin and PARP. These results show that Panax ginseng extract protects human neuronal SK-N-MC cells from the apoptosis induced by PCB 52. We suggest that Panax ginseng extracts may protect neuronal cells from oxidative injury.

In vivo effects of Panax ginseng extracts on the cytochrome P450-dependent monooxygenase system in the liver of 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed guinea pig.

The effects of the subchronic administration of Panax ginseng extracts were examined on the hepatic cytochrome P450-dependent monooxygenase system of guinea pigs pre-exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Panax ginseng extracts were intraperitoneally administered to guinea pigs at 100 mg/kg/day for 14 days from 1 week after a single intraperitoneal injection of 1 microg of TCDD/kg of body weight. TCDD treatment increased the total cytochrome P450 content 2.86-fold, and this was remarkably inhibited by the administration of Panax ginseng extracts. Treatment with ginseng extract alone also decreased the contents of cytochrome P450 by 33%, but both TCDD and ginseng extracts had no effect on cytochrome b(5) content. The administration of TCDD resulted in a 1.73-fold increase in microsomal NADPH-cytochrome P450 reductase activity in the guinea pig liver, and this was significantly inhibited by ginseng extracts, but treatment with ginseng extracts alone had no effect on its activity, and no statistical changes in the activity of NADPH-cytochrome b(5) reductase were observed in guinea pig liver due to TCDD and/or ginseng extract administration. Compared to the control, ECOD activity remarkably (1.76-fold) increased after TCDD administration, but this increase was completely inhibited by treatment with ginseng extract. Treatment with ginseng extract alone resulted in a 50% reduction of ECOD activity. TCDD administration remarkably induced benzphetamine demethylation (BPDM) activity, while ginseng extract also slightly increased the enzyme's activity, but the induction attributed to ginseng extracts was not statistically significant. Even though administration of ginseng extracts slightly inhibited TCDD-induced BPDM activity, the inhibition was not statistically significant. These results indicate that ginseng extract exerts different effect on the induction of P450 isozymes. From these results, we suggest that Panax ginseng extracts may act as an inhibitor of CYP1A rather than that of CYP2B.

α-Iso-cubebene exerts neuroprotective effects in amyloid beta stimulated microglia activation.

Schisandra chinensis is commonly used for food and as a traditional remedy for the treatment of neuronal disorders. However, it is unclear which component of S. chinensis is responsible for its neuropharmacological effects. To answer this question, we isolated α-iso-cubebene, a dibenzocyclooctadiene lignin, from S. chinensis and determined if it has any anti-neuroinflammatory and neuroprotective properties against amyloid ß-induced neuroinflammation in microglia. Microglia that are stimulated by amyloid ß increased their production of pro-inflammatory cytokines and chemokines, prostaglandin E2 (PGE2), nitric oxide (NO) and reactive oxygen species (ROS) and the enzymatic activity of matrix metalloproteinase 9 (MMP-9). We found this was all inhibited by α-iso-cubebene. Consistent with these results, α-iso-cubebene inhibited the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2) and MMP-9 in amyloid ß-stimulated microglia. Subsequent mechanistic studies revealed that α-iso-cubebene inhibited the phosphorylation and degradation of IκB-α, the phosphorylation and transactivity of NF-κB, and the phosphorylation of MAPK in amyloid ß-stimulated microglia. These results suggest that α-iso-cubebene impairs the amyloid ß-induced neuroinflammatory response of microglia by inhibiting the NF-κB and MAPK signaling pathways. Importantly, α-iso-cubebene can provide critical neuroprotection for primary cortical neurons against amyloid ß-stimulated microglia-mediated neurotoxicity. To the best of our knowledge, this is the first report showing that α-iso-cubebene can provide neuroprotection against, and influence neuroinflammation triggered by, amyloid ß activation of microglia.

In vivo Investigation of Anti-diabetic Properties of Ripe Onion Juice in Normal and Streptozotocin-induced Diabetic Rats.

The acute and subacute hypoglycemic and antihyperglycemic effects of drinkable ripe onion juice (Commercial product name is "Black Onion Extract") were investigated in normal and streptozotocin-induced diabetic rats. For tests of acute and subacute hypoglycemic effects, ripe onion juice (5 and 15 mL/kg b.w.) was administered by oral gavage to normal Sprague Dawley rats and measurements of fasting glucose levels and oral glucose tolerance tests were performed. Tolbutamide was used as a reference drug at a single oral dose of 250 mg/kg b.w. To test anti-hyper-glycemic activity, the ripe onion juice was administered to streptozotocin-induced diabetic rats by oral gavage at single dose of 15 mL/kg b.w. per day for 7 consecutive days. Oral administration of the ripe onion juice at either dosed level of 5 or 15 mL/kg b.w. showed no remarkable acute hypoglycemic effect in normal rats. The two dosed levels caused a relatively small reduction, only 18% and 12% (5 and 15 mL/kg b.w., respectively) decrease in glucose levels at 2 h after glucose loading in normal rats. However, at 3 h after glucose loading, blood glucose levels in the ripe onion juice-dosed rats were decreased to the corresponding blood glucose level in tolbutamide-dosed rats. Although showing weak hypoglycemic potential compared to that of tolbutamide, oral administration of ripe onion juice (15 mL/kg b.w.) for a short period (8 days) resulted in a slight reduction in the blood glucose levels that had elevated in Streptozotocin-induced diabetic rats. In conclusion, these results suggest that the commercial product "Black Onion Extract" may possess anti-hyperglycemic potential in diabetes.

A novel Helicosporium isolate and its antimicrobial and cytotoxic pigment.

One Helicosporium strain, isolated from a wilted chestnut tree, evidenced in vitro antimicrobial activity against various types of bacteria and fungi, and generated a diffusible pigment. The antimicrobial compounds and the diffusible pigment of the Helicosporium sp. isolate were purified via solvent fractionation, column chromatography, and recycling preparative chromatography. Both the major antimicrobial compound and the diffusible pigment were identified as 2-methylresorcinol via nuclear magnetic resonance spectroscopy. Therefore, 2-methylresorcinol, a diffusible pigment generated by Helicosporium sp., appears to be an active antimicrobial principle. This pigment also exhibited considerable cytotoxicity against mammalian cells.

Identification of 5-hydroxy-3,6,7,8,3´,4´-hexamethoxyflavone from Hizikia fusiforme involved in the induction of the apoptosis mediators in human AGS carcinoma cells.

An 80% ethanol extract of Hizikia fusiforme was obtained and followed by successive fractionation using the organic solvents n-hexane, ethyl acetate, and n-butanol to identify the antioxidative substance. The aqueous part of the nbutanol fractionation step, showing high antioxidative activity, was subjected to reverse-phase liquid chromatography. As a result, a substance purified from a BB-2 fraction showed high antioxidative activity. The m/z 419 [M+H] molecular ion peak in the fraction was observed by the analysis of the ESI-LC/MS spectrum. By the analysis of 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) spectra, a unique compound of the fraction was biochemically identified as a 5-hydroxy-3,6,7,8,3´,4´- hexamethoxyflavone (5HHMF). We also investigated the effect of 5HHMF on human gastric AGS carcinoma cells. Western blot analysis suggested that the flavone substantially increased the levels of the death receptor-associated apoptosis mediators Fas, Fas L, FADD, TRADD, and DR4 in a concentration-dependent manner. The levels of Fas, Fas L, TRADD, and DR4 in the cells treated with 5HHMF (5 microgram/ml) were approximately 26.4-, 12.8-, 6.7-, and 9.8- times higher than those of non-treated cells, respectively. Of note, the level of FADD protein in the cells exposed to 5HHMF (1 microgram/ml) increased approximately 9.6-times. In addition, the cleavage of caspase-3, -8, and -9 in cultured AGS cells treated with 5HHMF was significantly confirmed. Therefore, our results suggest that 5HHMF from H. fusiforme is involved in the induction of death receptor-associated apoptosis mediators in human gastric AGS carcinoma cells.

Detection of a unique fibrinolytic enzyme in Aeromonas sp. JH1.

A fibrinolytic enzyme was found in a Gram-negative bacterium, Aeromonas sp. JH1. SDS-PAGE and fibrinzymography showed that it was a 36 kDa, monomeric protein. Of note, the enzyme was highly specific for fibrinogen molecules and the hydrolysis rate of fibrinogen subunits was highest for α, ß, and γ chains in that order. The first 15 amino acids of N-terminal sequence were X-D-A-T-G-P-G-G-N-V-X-T-G-K-Y, which was distinguishable from other fibrinolytic enzymes. The optimum pH and temperature of the enzyme were approximately 8.0 and 40°C, respectively. Therefore, these results provide a fibrinolytic enzyme with potent thrombolytic activity from the Aeromonas genus.

Purification and biochemical characterization of a 17 kDa fibrinolytic enzyme from Schizophyllum commune.

A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods, including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.

Characterization of Pseudomonas sp. BCNU 171 tolerant to organic solvents.

An organic solvent-tolerant bacterium, designated as Pseudomonas sp. BCNU 171, was isolated from industrial wastewater in Korea, on the basis of its ability to survive in the presence of benzene, toluene, propylbenzene and xylenes. Its tolerance limits were 8 mM in phenol, 20 mM in benzene and 60 M in toluene. The log P value of phenol was approximately 1.5, which indicates that Pseudomonas sp. BCNU 171 exhibits the highest tolerance to organic solvents. Pseudomonas sp. BCNU 171, a relative of P. putida, P. mosselii and P. moteillii based on phylogenetic analyses using 16S rRNA sequences, was designated as a new sp. that is tolerant to a wide spectrum of organic solvents, especially xylene isomers. These findings may facilitate the understanding of organic solvent tolerance in bacterial cells.

Purification of a fibrinolytic enzyme (myulchikinase) from pickled anchovy and its cytotoxicity to the tumor cell lines.

A fibrinolytic enzyme, myulchikinase, from a Korean seasoning ingredient, myul-chi-jeot-gal, has been purified to electrophoretic homogeneity. The molecular mass of the myulchikinase was estimated to about 28 kDa by SDS-PAGE and gel filtration. Amino acid sequence of the NH2-terminal of myulchikinase showed significant homology with other fibrinolytic enzymes including trypsin from starfish, katsuwokinase, and rat pancreatic elastase II. The purified myulchikinase hydrolyzed various synthetic substrates with different substrate specificity and cytotoxic to the tumor cell lines.

A one-pot synthesis of pyrido[2,3-b][1,4]oxazin-2-ones.

Pyrido[2,3-b][1,4]oxazin-2-ones are conveniently prepared in excellent yields by a one-pot annulation of N-substituted-2-chloroacetamides with 2-halo-3-hydroxypyridines with use of cesium carbonate in refluxing acetonitrile. The key transformation features a Smiles rearrangement of the initial O-alkylation product and subsequent cyclization.